The term "serum" as used herein means the liquid portion of the blood that remains after blood cells and fibrinogen/fibrin are removed. The term "plasma" as used herein means the liquid portion of blood that remains after blood cells are removed. The term "serum products" means any component of the serum, such as soluble proteins, lipids or carbohydrates or combinations thereof. An example of a serum protein is albumin.
The use of freezing techniques to freeze various cells and tissues for long term storage has become increasingly popular. In particular, the agricultural industry has successfully utilized these freezing techniques in animal husbandry. Furthermore, freezing techniques have been used to preserve ova, sperm and embryos from humans for later use.
A large industry is developing that is concerned with methods of fertilizing and transferring ova and embryos to surrogate females. The advantages of this technology to the animal husbandry industry include increasing the reproductive rate of valuable animals, decreasing the generation interval, increasing the number of progeny per female through controlled multiple births, and transporting embryos with selected genetic characteristics to distant places. This technology has been used for over 300 species including, but not limited to, cattle, swine, sheep, goats and other agriculture animals. The technology has also been used for freezing human embryos.
By way of example, a brief description of the biology of bovine reproduction follows: Bovine embryos move from the oviduct to the uterus four to five days after estrus (three to four days after ovulation), although in super ovulated cows, a few remain in the oviduct through day seven. A high percentage of embryos can usually be recovered nonsurgically from the uterus six or more days after the beginning of estrus.
Several procedures are currently available for recovering embryos. One such procedure comprises inserting a Rusch or Foley catheter through the cervix into the uterus by palpating through the wall of the rectum with one hand as is done for artificial insemination. The latex catheter usually consists of three channels for inflow, outflow, and inflation of the balloon-like cuff that prevents the escape of fluid after insertion. Each uterine horn is filled and emptied several times with 30-200 ml of fluid each time according to the size of the uterus. The embryos are flushed out with this fluid into large graduated cylinders. Embryos can be filtered with a 50-70.mu. mesh filter or allowed to settle for thirty minutes and can then be located under a stereo microscope by searching through an aliquot from the bottom of the cylinder. They are then stored in small containers or loaded into 0.25 to 5 ml straws until transferred.
In addition, the embryos can be recovered surgically. This is the procedure of choice for laboratory animals and for certain agricultural animals and may be the method of individual operator preference for large agricultural animals.
Embryos from the one cell to the early blastocyst stage (7 to 8 days after estrus) are between 120 and 140 micrometers in diameter exclusive of the zone of pellucida. Between days 8 and 10, they double in diameter, hatch from the zone of pellucida, and then grow to 20 cm or more in length by day 18. Since bovine embryos form no intimate attachment to the uterus before day 18, they can be recovered nonsurgically until this time, although they are increasingly prone to damage after day 14. It appears that a larger number of normal embryos can be obtained nonsurgically 6 to 8 days after estrus than at other times. (See U.S. Pat. No. 4,780,451.)
After the embryos are collected from the donor cow, they should be transfered to recipient animals within a short period of time. Alternatively, the embryos can be cooled to refrigerator temperatures or frozen. Conventional freezing media contain some type of serum. For example, newborn calf serum or bovine serum albumin (BSA) plus additional glycerol and/or other constituents have been widely used in media for embryo freezing as both cryoprotectants and surfactants.
However, these sera have a major problem in that they can transfer viral contaminants to the embryo including, but not limited to, bovine virus diarrhea (BVD), infectious bovine rhinotrachitis (IBR), bovine spongiform encephalopathy (BSE), Scrapie, blue tongue, and foot and mouth disease. In addition, serum from animals inherently adds undefined components to the freezing media allowing for the possibility of deleterious effects on the embryos.
There is accordingly a need for a composition and method for culturing and freezing living tissues and cells, including embryos, which do not contain serum or serum products from animals. There is also a need for a culturing and freezing medium which is completely defined and does not contain any serum or serum products from animals or humans. The freezing medium should protect the living tissue or cells that are being frozen and allow high viability of the cells or tissue when they are thawed.